Virtual Laboratory: Cellular Laboratory

ELISPOT: Summary; Science; Clinical

Science

The ELISPOT test is an ‘in vitro’ assay, this means that it recreates an actual biological process in a controlled laboratory environment. In the case of TB diagnosis, we are aiming to recreate an immune response against the TB bacterium that would occur only in people that have a current infection or have had an infection in the past.
A spinning process called centrifugation separates the lymphocytes (cells responsible for recognition of pathogenic bacteria) from a patient’s blood. These separated cells are mixed with a solution containing specific bacterial proteins (antigens) and incubated for 16-20 hours in a special multi well plate at 37ºC (body temperature). The bottoms of the wells are coated with antibodies that specifically capture a protein called IFN-γ that is released by lymphocytes when they recognise molecules as ‘foreign’. If a patient has been previously exposed to TB, they will have generated a population of lymphocytes that recognise and that are activated by the antigens present in the well. When these cells are activated, they release IFN-γ which becomes trapped by the specific antibodies that are bound to the plate. After the incubation, the cells and antigen mixes are washed off and another antibody, this time one attached to an enzyme, is applied that binds to the trapped IFN-y. After a short incubation, the excess antibody is washed off and then a substance called a substrate is added. The enzymes attached to the antibody cleave the substrate and a coloured precipitate is formed. The final effect is a well that has a number of coloured areas, or “spots” on its base. Each spot indicates the footprint of a cell that has been activated by the TB antigens. The presence of these spots is a strong indication of an active or latent TB infection. Because the outcome of this test could have serious implications for a patient the results are double-checked by the scientist performing the test and by a scientific or medial consultant.